The proposed studies will investigate nitric oxide (NO)-mediated phosphorylation mechanisms that result in hypoxic neuronal death by transcription-independent and transcription-dependent pathways in the newborn. We propose that NO produced during hypoxia leads to increased phosphorylation of the cell death repressor protein Bcl-2 and cyclic AMP-response element binding (CREB) protein. Phosphorylation of Bcl-2 and CREB proteins will correlate with the severity of cerebral tissue hypoxia. We propose that NO free radicals generated during hypoxia lead to inactivation of protein tyrosine phosphatase (PIP) and mitogen-activated kinase phosphatase1 (MKP-1) and MKP-3. Inactivation of these phosphatases results in increased phosphorylation of BcI-2, a NO-mediated transcription-independent mechanism of hypoxic neuronal death. Furthermore, we propose that NO generated during hypoxia increases intranuclear Ca++-influx in neuronal nuclei leading to activation of nuclear CaM-kinase resulting in increased phosphorylation of CREB protein and increased expression of the cell death promoter protein Bax, a NO-mediated transcription-dependent mechanism of neuronal death following hypoxia. The degree of brain hypoxia in vivo will be monitored by continuous measurement of high energy phosphate compounds with 31P-nuclear magnetic resonance spectroscopy and confirmed biochemically. Experimental protocols will be carried out on newborn piglets investigating: (1) the relationship of quantitative tissue hypoxia to phosphorylation of Bcl-2 and CREB proteins, (2) the effect of hypoxia on: (a) the activity and expression of SH-PTP-1, MKP-1 and MKP-3, (b) the activation and activity of MAP kinases ERK and JNK; (3) the effect of hypoxia on nitrosylation of cysteine residues in SH-PTP-1, MKP-1 and MKP-3; (4) the relationship of NO-mediated nitrosylation of cysteine residues in vitro to inhibition of the activity of protein tyrosine phosphatase and MAP kinase phosphatases MKP-1 and MKP-3; (5) the effect of NO donors on Ca++-influx in neuronal nuclei; (6) the effect of hypoxia on the activity of neuronal nuclear CaM-kinase and subsequent expression of cell death promoter protein Bax; and (7) the effect of administration of nitric oxide synthase (NOS) inhibitors during hypoxia on activity of phosphatases, phosphorylation of Bcl-2 and CREB, expression of Bax and immunocytochemical and morphological indices of neuronal death. These studies will provide a novel mechanism of NO-mediated phosphorylation of specific proteins resulting in hypoxic neuronal death by transcription-independent and transcription-dependent pathways. The elucidation of molecular mechanisms of NO-mediated phosphorylation of the cell death repressor protein Bcl-2 and expression of the cell death promoter protein Bax in response to hypoxia will aid in the development of novel preventive strategies for hypoxia-induced brain dysfunction in the newborn.